This is a competitive renewal of NIH Grant DK31036-13 which has focused on the structure and turnover of the insulin receptor. During the past 5 years considerable progress has been made in characterization of the structure and function of the receptor, the pathways of turnover of the receptor, the structure of the insulin receptor gene, and the nature of insulin receptor signaling. As a result 40 papers have been published, or are in press or preparation. There remain, however, a number of important, unanswered questions concerning the basic features of the receptor, including how insulin binding the alpha-subunit results in stimulation of kinase activity in the beta-subunit, how the insulin proreceptor is processed, and perhaps most importantly, can one develop more physiological models to study the effect of insulin receptor stimulation or the lack thereof. During the next 5 years, we will focus on both more short-term directed experiments to answer these specific questions, as well as the more long-term need to develop appropriate model systems for evaluation of receptor function. Specifically, we will determine the mechanism by which transmembrane mutations in the receptor result in constitutive activation or loss of insulin-stimulated activation of the receptor using both in vivo and in vitro reconstitution systems. We will study the effect of chronic receptor activation in the absence of insulin and mechanisms of receptor mediated desensitization using the constitutively activated receptor in cell culture models, as well as in whole animals with a transgenic approach. We will also develop an anchored insulin receptor-IRS-1 chimeric molecule to determine the role of differential receptor and substrate trafficking in insulin signaling. To define the enzymes involved in proreceptor processing, the nature of the enzymatic recognition site and the cellular sites of receptor processing we will use in vitro assays with both the holoreceptor and a peptide library approach. In addition, we will put long-term efforts into development of more physiological models of insulin receptor function. This will be accomplished in cultured cells using retroviral vectors and in intact animals using transgenic approaches, especially tissue specific approaches. Finally we will attempt to develop an insulin receptor deficient mouse, including tissue specific receptor deficiency, using targeted gene knockout by homologous recombination, and to explore methods for tissue specific reconstitution.